Bacterial topoisomerase i inhibitors with antibacterial activity

ABSTRACT

The present invention provides compounds as bacterial topoisomerase inhibitors with antibacterial activity. The present invention also provides pharmaceutical compositions comprising at least one of the compounds and methods of using the compounds and pharmaceutical compositions as antibacterial agents for treating infectious diseases,

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/707,537, filed Sep. 18, 2017, which claims the priority benefit ofU.S. Provisional Application Ser. No. 62/395,652, filed Sep. 16, 2016,both of which are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under AI069313, GM103466and AI092315 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF INVENTION

Microbial pathogens are becoming increasingly resistant to currentantibiotics, limiting the availability of clinical treatment options forbacterial infections (1). It is imperative to develop novel classes ofantibacterial compounds, preferably against a new target, to avoidcross-resistance. Tuberculosis (TB) infects 9.6 million people a yearand causes 1.5 million deaths each year (2). The problem presented bymulti-drug resistance is illustrated by the 480,000 cases of multi-drugresistant TB (MDR-TB) that do not respond to first line treatment drugs,with around ten percent of these cases being extensively-drug resistanttuberculosis (XDR-TB) that are resistant to even some of the second linedrugs (2, 3). New combinations of anti-TB drugs are needed to treat theMDR-TB and XDR-TB cases.

Topoisomerases are needed in every organism to regulate DNA topology sothat vital cellular processes including DNA replication, transcription,recombination and repair can proceed without hindrance (4, 5). Type IIAtopoisomerases cut and rejoin a double strand of DNA during catalysis(6). DNA gyrase and topoisomerase IV are prokaryotic type IIAtopoisomerases that have been extensively explored as validated targetsfor antibacterial therapy in the clinic (7, 8). At least one type IAtopoisomerase is present in every bacterial pathogen to resolvetopological barriers that require the cutting and rejoining of a singlestrand of DNA and passage of DNA through the transient break (9).Topoisomerase I is the major type IA topoisomerase activity responsiblefor preventing excessive negative supercoiling in bacteria (10, 11).

Bacterial topoisomerase has received some recent interest as a novelantibacterial drug target (9, 12). Poison inhibitors of topoisomeraseenzymes can lead to the accumulation of the intermediatetopoisomerase-DNA cleavage complex and subsequently result in bacterialor cancer cell death. Escherichia coli topoisomerase I (EcTopI) is themost extensively studied type IA topoisomerase, with crystal structuresof covalent cleavage complex (13) and full-length enzyme-DNA complex(14) available. Inhibition of EcTopI by endogenous polypeptideinhibitors (15-17) can lead to cell killing even though compensatorymutations could allow E. coli strains with topA deletion to be viable(18, 19). There is also evidence that topoisomerase I function isessential for a number of bacterial pathogens including Streptococcuspneumoniae (20) and Helicobacter pylori (21). There is only one type IAtopoisomerase encoded by the genomes of Mycobacteria. Mycobacteriumtuberculosis topoisomerase I (MtbTopI) has been demonstrated in geneticstudies to be essential for viability both in vitro (22, 23) and in vivo(23). Experimental data showed that the minimal inhibitoryconcentrations (MICs) of select small molecules against Mycobacteriumtuberculosis can be shifted by overexpression of topoisomerase I (23,24), further validating topoisomerase I as a vulnerable target in M.tuberculosis for chemical inhibition.

Clinically, topoisomerase enzymes represent attractive and successfultargets for anticancer and antibacterial chemotherapy. Many of the smallmolecules identified previously as bacterial topoisomerase I inhibitorsare DNA intercalators (20, 24-26) or minor groove binders (27, 28) thatwould not be attractive candidates for antibiotics development.Therefore, there is an urgent need to develop compounds that targetbacterial pathogens, in particular, through the inhibition of bacterialtopoisomerase I.

BRIEF SUMMARY

The current invention provides compounds and methods for inhibiting theactivity of topoisomerase. These compounds and methods according to thecurrent invention can further be used to control bacterial pathogens.The current invention also provides pharmaceutical compositionscomprising one or more compounds of the current invention, and methodscomprising administration of the compositions for treating a surface ora subject infected with a bacterial pathogen.

In one embodiment, the compound comprises a scaffold having a generalstructure of (I):

wherein R₁, R₂, R₃ and R₄ are each independent groups, wherein R₁, R₃and R₄ are each independently selected from H, —OH, —NH₂, —NO₂, —NHMe,acetyl group (—Ac), —CN, —NHAc, —NHCH₂CH₂NH₂, —CF₃, fluorine, chloride,bromine, iodine, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heterocycloalkyl, substitutedheterocycloalkyl, amino group, and substituted amino group; and R₂ isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, and substituted cycloalkenyl.

In a further embodiment, R₁ is selected from —NH₂, —NO₂, —Ac, —CN, —NHAcand —CF₃; R₂ is H or an ethyl group; R₃ is an amino group, substitutedamino group including amine group with lipophilic side chainsor selectedfrom:

wherein R₅ is H, OH, Me, NH₂, NO₂, tert-butyloxycarbonyl (Boc) amine(e.g., NHBoc), fluorine, chloride, bromine, iodine, alkyl, orsubstituted alkyl.

In one embodiment, the compounds have activity against bacterialpathogens, including both gram-positive and negative bacteria. In afurther embodiment, the compounds have activity against mycobacteria. Inanother further embodiment, the compounds have activity against E. coli,Staphylococcus aureus, Streptococcus pneumoniae, Helicobacter pylori,Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti,Mycobacterium canetti, Mycobacterium smegmatis and/or Mycobacteriumtuberculosis. In a preferred embodiment, the compounds have activityagainst M. tuberculosis.

In other embodiments, the compounds have activity againstnon-tuberculosis mycobacteria (NTM), e.g., Mycobacterium avium complex(MAC), Mycobacterium kansasii, and Mycobacterium abscessus, and thus,can be used to treat NTM infection in a subject. NTM are all the othermycobacteria which can cause pulmonary disease. NTM can also infectskin, soft tissues and lungs of cystic fibrosis patients.

In one embodiment, the compounds are used for treatment of infections inthe form of biofilms formed by mycobacteria, including TB and NTM. Suchbiofilms are often difficult to treat with antibiotics.

In another embodiment, the compounds have activity against drugresistant bacterial pathogens, preferably, M. tuberculosis andStaphylococcus aureus, more preferably, Methicillin-resistantStaphylococcus aureus (MRSA).

In one embodiment, the compounds inhibit the activity of topoisomerase,preferably, the type IA family of topoisomerase, more preferably,bacterial topoisomerase I. Additionally, the compounds exhibit selectiveinhibition of bacterial topoisomerase I over DNA gyrase.

In one embodiment, the compounds target bacterial pathogens through theinhibition of topoisomerase. In a further embodiment, the compoundsinhibit the growth of bacterial pathogens by targeting the type IAfamily of topoisomerase. In a preferred embodiment, the compoundsexhibit cytotoxicity by inhibiting bacterial topoisomerase I. In a morepreferred embodiment, the compounds inhibit M. tuberculosistopoisomerase I (MtbTopI).

In one embodiment, the compounds are bactericidal against bacterialpathogens, including both gram-positive and negative bacteria. In afurther embodiment, the compounds are bactericidal against mycobacteria.In another further embodiment, the compounds are bactericidal against E.coli, Staphylococcus aureus, Streptococcus pneumoniae, Helicobacterpylori, M. smegmatis and/or M. tuberculosis, preferably, M.tuberculosis.

In another embodiment, the compounds are bactericidal against drugresistant bacterial pathogens, preferably, M. tuberculosis andStaphylococcus aureus, more preferably, Methicillin-resistantStaphylococcus aureus (MRSA).

In one embodiment, the compounds are used as antibacterial drugs inantibacterial therapy. In a specific embodiment, the compounds are usedin treatment of infectious diseases, preferably, tuberculosis.

In one embodiment, the compounds can be used as antituberculosis agents.

In one embodiment, the current invention provides a pharmaceuticalcomposition comprising one or more compounds of the subject invention.The composition can further comprise a pharmaceutically acceptablecarrier.

In a further embodiment, the compounds are in a pharmaceuticallyacceptable salt form or a form of free base. The composition may furthercontain pharmaceutically acceptable ingredients including metal saltsand/or buffers. In certain embodiments, the pharmaceutical compositionscan also include additional pharmaceutically active compounds know inthe art.

In one embodiment, the current invention provides a pharmaceuticalcomposition for treating conditions involving bacterial infection,preferably tuberculosis.

In one embodiment, the current invention also provides a methodcomprising the administration of an effective amount of thepharmaceutical composition for treating a subject infected with abacterial pathogen. In a preferred embodiment, the subject is a human.In another preferred embodiment, the human patient is infected with M.tuberculosis and/or MRSA.

In one embodiment, the current invention provides a method for treatingTuberculosis, preferably, drug resistant tuberculosis.

The pharmaceutical composition can be administered through, for example,oral, rectal, bronchial, nasal, topical, buccal, sub-lingual,transdermal, vaginal, intramuscular, intraperitoneal, intravenous,intra-arterial, intracerebral, and interaocular administration.

The present invention also provides a method of inhibiting type IAtopoisomerase in a subject, preferably in a human or a bacterium,comprising the administration of one or more of the compounds to thehuman or bacterium.

The present invention also provides a kit comprising the compounds orpharmaceutical compositions as described herein.

The compounds, compositions, methods and kits described herein can beused in connection with pharmaceutical, medical, veterinary, anddisinfection applications, as well as fundamental biological researchand methodologies, as would be identified by a skilled person uponreading the present disclosure.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Chemical structures of clinical fluoroquinolone antibiotics andfluoroquinophenzoxazine derivatives including topo I inhibitor 1.

FIG. 2. Scheme 1. Synthesis of 9-amino-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid (1).

FIG. 3. Scheme 2. Synthesis of fluoroquinophenoxazine derivatives 7b-e.

FIG. 4. Scheme 3. Synthesis of diverse amino-substitutedfluoroquinophenoxazines 9-11 and acetyl-protected quinophenoxazine 12.

FIG. 5. Scheme 4. Synthesis of fluoroquinophenoxazine chiral aminederivatives 16 and 17.

FIGS. 6A-6B. Inhibition of E. coli topoisomerase relaxation activity byrepresentative 11a and 11b. A) E. coli topoisomerase I inhibition assayswith supercoiled plasmid DNA. B) E. coli DNA gyrase inhibition assayswith relaxed plasmid DNA. C refers to DMSO control; Nor refers toNorfloxacin (125 μM) control; S refers to supercoiled plasmid DNA; Nrefers to Nicked DNA; FR refers to fully relaxed DNA; PR refers topartially relaxed DNA.

FIG. 7. The correlation chart of experimental versus predicted valuesfor the training and test set compounds.

FIGS. 8A-8D. Representative CoMFA steric and electrostatic contour maps.A). Steric contour maps with 6c; B). Steric contour maps with 11 b; C).Electrostatic contour maps with 6c; and D) Electrostatic contour mapswith 11b.

DETAILED DISCLOSURE

The current invention provides compounds and methods for inhibiting theactivity of topoisomerase. The compounds according to the invention haveactivity against one or more bacterial pathogens. The current inventionalso provides a pharmaceutical composition comprising at least one ofthe compounds, and methods comprising administration of the compositionsfor treating a subject infected with a bacterial pathogen or in need ofsuch administration for inhibiting the activity of topoisomerase.

In one embodiment, the compound comprises a scaffold having a generalstructure of (I):

wherein R₁, R₂, R₃ and R₄ are each independent groups, wherein R₁, R₃and R₄ are each independently selected from H, —OH, —NH₂, —NO₂, —NHMe,—Ac, —CN, —NHAc, —NHCH₂CH₂NH₂, —CF₃, fluorine, chloride, bromine,iodine, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heterocycloalkyl, substitutedheterocycloalkyl, amino group, and substituted amino group; R₂ isselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, and substituted cycloalkenyl.

In another embodiment, R₁, R₂, R₃ and R₄ are each independent groups,wherein R₁ is selected from H, —OH, —NH₂, —NO₂, —NHMe, —Ac, —CN, —NHAc,—NHCH₂CH₂NH₂, and —CF₃, R₂ is an H or an alkyl group; and R₃ is selectedfrom fluorine, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heterocycloalkyl, substitutedheterocycloalkyl, amino group, and substituted amino group withdifferent functionalities such as piperazine, 1-methylpiperazine, andmorpholine; and R₄ is selected from H, —OH, —NH₂, —NO₂, —Ac, —CN, —NHAc,—NHCH₂CH₂NH₂, —CF₃, fluorine, chloride, bromine, iodine, and alkyl.

In a further embodiment, R₁ selected from —NH₂, —NO₂, —Ac, —CN, and—CF₃.

In one embodiment, each R group may comprise other functional groupsincluding quinolones, indoles, benzofurans, benzothiophenes, andbiphenyls.

In a specific embodiment, each of the R groups may comprise a positivelycharged functional group, or a large aromatic group. Furthermore, eachof the R groups may comprise a methyl naphthyl group wherein thenaphthyl group includes but is not limited to dihydroxyphenyl,halogenated phenyls, aliphatic groups.

In one embodiment, the R groups may each independently include alkylamines with varying chain lengths, cyclic amines (e.g., piperazine suchas 1-methylpiperazine, morpholine, piperidine), cyclic alkyls, and arylgroups.

In a further embodiment, R₂ is H or an ethyl group. R₃ is an aminogroup, substituted amino group including amine group with lipophilicside chains or is selected from:

wherein R₅ is —H, —OH, —Me, —NH₂, —NO₂, —NHBoc, —Ac, —CN, —NHAc,—NHCH₂CH₂NH₂, —CF₃, fluorine, chloride, bromine, iodine, alkyl, orsubstituted alkyl.

In certain embodiments, each of R₁, R₂, R₃ and R₄ is selected from:

wherein n is at least 2, preferably ranging from 2 to 5.

In certain embodiments, each of R₁, R₂, R₃ and R₄ is independentlyselected from:

andwherein Z is preferably O, NH, NMe, or S.

In one embodiment, the compound is:

In one embodiment, the compound is selected from:

In one embodiment, the compound is selected from:

In one embodiment, the compound is selected from:

In one embodiment, the compound is:

wherein R′ is selected from:

In one embodiment, the compound is selected from:

In one embodiment, the compounds are fluoroquinophenzoxazine andderivatives thereof.

In one embodiment, certain compounds of the present disclosure possessasymmetric carbon atoms (optical or chiral centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids,and individual isomers are encompassed within the scope of the presentdisclosure. The present disclosure is meant to include compounds inracemic and optically pure forms. Optically active (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. When the compoundsdescribed herein contain olefenic bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

Unless otherwise stated, structures depicted herein also are meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Chemical Definitions

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability to change onefunctional group for another functional group provided that the valencyof all atoms is maintained. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. The substituents also may be furthersubstituted (e.g., an aryl group substituent may have anothersubstituent off it, such as an alkyl group, which is furthersubstituted, for example, with fluorine at one or more positions).

Where a group may be substituted by one or more of a number ofsubstituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds that are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). In particular embodiments, theterm “alkyl” refers to C₁₋₂₀ inclusive, linear (i.e., “straight-chain”),branched, or cyclic, saturated or at least partially and in some casesfully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicalsderived from a hydrocarbon moiety containing between one and twentycarbon atoms by removal of a single hydrogen atom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having I to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂₅—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR, and/or —SO₂R′.Where “heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl,” as well as their divalent derivatives) are meant toinclude both substituted and unsubstituted forms of the indicated group.Optional substituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2 m′+1), where m′ is the total number of carbon atoms in suchgroups. R′, R″, R′″ and R″″ each may independently refer to hydrogen,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. As used herein, an “alkoxy” group is an alkyl attachedto the remainder of the molecule through a divalent oxygen. When acompound of the disclosure includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. When R′and R″ are attached to the same nitrogen atom, they can be combined withthe nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:(A) —OH, —NH₂, —SH, —CN,—CF₃, —NO₂, oxo, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,substituted with at least one substituent selected from: (i) oxo, —OH,—NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,substituted with at least one substituent selected from: (a) oxo, —OH,—NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl,substituted with at least one substituent selected from oxo, —OH, —NH₂,—SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described hereinabove fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

Compound Activities

In one embodiment, the compounds according to the current inventiontarget topoisomerase, preferably, bacterial topoisomerase, morepreferably bacterial topoisomerase in the IA family, and mostpreferably, bacterial topoisomerase I such as EcTopI and MtbTopI.

In a further embodiment, the compounds inhibit the activity oftopoisomerase, preferably, the type IA family of topoisomerase, morepreferably, bacterial topoisomerase I such as EcTopI and MtbTopI.Additionally, in preferred embodiments, the compounds exhibit selectiveinhibition of bacterial topoisomerase I over DNA gyrase.

In one embodiment, the compounds have activity against bacterialpathogens, including both gram-positive and negative bacteria. In afurther embodiment, the compounds have activity against mycobacteria. Inanother further embodiment, the compounds have activity against E. coli,Staphylococcus aureus, Streptococcus pneumoniae, Helicobacter pylori,Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti,Mycobacterium canetti, M. smegmatis and/or M. tuberculosis, preferably,M. tuberculosis.

In another embodiment, the compounds have activity against drugresistant bacterial pathogens, preferably, M. tuberculosis and/orStaphylococcus aureus.

In other embodiments, the compounds have activity againstnon-tuberculosis mycobacteria (NTM), e.g., Mycobacterium avium complex(MAC), Mycobacterium kansasii, and Mycobacterium abscessus, and thus,can be used to treat NTM infection in a subject. NTM are all the othermycobacteria that can cause pulmonary disease. NTM can also infect skin,soft tissues and lungs of cystic fibrosis patients.

In one embodiment, the compounds are used for treatment of infections inthe form of biofilms formed by mycobacteria, including TB and NTM. Suchbiofilms are often difficult to treat with antibiotics.

In one embodiment, the compounds inhibit the growth of bacterialpathogens, preferably, through the inhibition of topoisomerase, morepreferably, through the inhibition of the type IA family oftopoisomerase. In a preferred embodiment, the compounds are bactericidalby inhibiting bacterial topoisomerase I, preferably, MtbTopI.

In one embodiment, the compound NSC648059 (1) has a low micromolarinhibitory activity (IC₅₀=0.8-2.0 μM) against Escherichia colitopoisomerase I. Structurally, 1 is a fluoroquinophenzoxazine derivativewith a unique planar tetracyclic ring system, belonging to a member ofan extended chemical class of fluoroquinolone antibiotics. In theclinic, fluoroquinolones including norfloxacin and ciprofloxacin(FIG. 1) represent some of the most successful antibiotic classes, whosemechanisms of action are to inhibit bacterial DNA gyrase andtopoisomerase IV as well as relaxation of supercoiled DNA and thus topromote breakage of double-stranded DNA [29]. Specifically,fluoroquinophenoxazines such as A-62176 and A-85226 (FIG. 1) have beenreported as antibacterial [30, 31] and anticancer [32-35] agents. Forexample, A-62176 exhibited good activity against several cancer celllines with IC₅₀ values ranging from 0.87-4.34 μM [32].

The growth inhibition IC₅₀ refers to the minimum compound concentrationthat inhibits the growth of bacteria, preferably, growth of M.tuberculosis, in comparison to a control in the absence of any compoundsby 50%.

In one embodiment, the compounds act as the inhibitor of bacterialtopoisomerase, preferably, topoisomerase I by interacting with theenzyme alone to prevent DNA binding or by interacting with theenzyme-DNA complex to inhibit enzyme function and/or DNA cleavage and/orDNA religation. In a further embodiment, the compounds also targetbacterial topoisomerase I with different substitutions/mutations locatedin the conserved or non-conserved sequences or motif. In a preferredembodiment, the compounds bind to the enzyme-DNA complex to form adrug-enzyme-DNA ternary structure.

In certain embodiments, the compounds exhibit antibacterial activity byperturbing the interaction of bacterial topoisomerase I, in particular,EcTopI and MtbTopI with other cellular components such as RNApolymerase, which further leads to increased susceptibility toantibacterial compounds, and reduced tolerance to challenges such ashigh temperature, acids, and oxidative stress.

In some embodiments, the compounds may be derived from the scaffold (I)or analogs of the scaffold (I). The compounds and analogs may displaydifferent selectivity, target specificity, binding affinity, cellpenetration and retention properties while inhibiting the activity oftopoisomerase, preferably, bacterial topoisomerase I, as well asinhibiting the growth of bacteria, preferably, mycobacteria, morepreferably, M. tuberculosis. The use of mycobacteria strains withdifferent levels of topoisomerase I expression in cell based-assayscould be used to complement the enzyme-based assays to identify andoptimize other analogues that can target topoisomerase I. The compoundscan also be identified with other assays such as multi-stress in vitrodormancy assay and/ or luminescent assay.

In one embodiment, the compounds are bactericidal and/or bacteriostaticagainst bacterial pathogens, including both gram-positive and negativebacteria. The compounds are effective in eliminating bacterial pathogensunder all growth condition. In a further embodiment, the compounds arebactericidal against mycobacteria. In another further embodiment, thecompounds are bactericidal against E. coli, Staphylococcus aureus,Streptococcus pneumoniae, Helicobacter pylori, M. smegmatis or M.tuberculosis, preferably, M. tuberculosis, and MRSA.

In another embodiment, the compounds are bactericidal against drugresistant bacterial pathogens, preferably, M. tuberculosis andStaphylococcus aureus.

In other embodiments, the compounds are bactericidal and/orbacteriostatic against non-tuberculosis mycobacteria (NTM), e.g.,Mycobacterium avium complex (MAC), Mycobacterium kansasii, andMycobacterium abscessus, and thus, can be used to kill NTM in a subject.

In one embodiment, the compounds are used for treatment of infections inthe form of biofilms formed by mycobacteria, including TB and NTM. Suchbiofilms are often difficult to treat with antibiotics.

In one embodiment, the compounds are used as antibacterial drugs ingeneral or pathogen specific therapy. The compounds according to thecurrent invention can be used in treatment of infectious diseases,preferably, Tuberculosis. In some embodiments, the compounds can be usedin combination with other drugs for infectious diseases to achievesynergistic effects for overcoming the resistance problem and reducingtime required for treatment. Preferably, the infectious disease isTuberculosis.

In one embodiment, the molecular scaffold can lead to compounds thatrepresent a new class of bactericidal antimycobacteria agents withtopoisomerase I being involved in the cellular mode of action.

In one embodiment, the present invention provides the compounds andsalts and derivatives thereof. Derivatives of the compounds include anypharmaceutically acceptable ester, salt of an ester, alcohol, diol,ether, aldehyde, ketone, carboxylic acid or enol of a compound that canbe made from the compounds by a chemical or physical process. Thecompounds may be in a purified form.

Pharmaceutical Composition

In one embodiment, the current invention provides a pharmaceuticalcomposition comprising one or more of the compounds. The compositionfurther comprises a pharmaceutically acceptable carrier. In certainembodiments, the pharmaceutical compositions can also include additionalpharmaceutically active compounds know in the art. One or moreadditional antibiotics may also be included in the composition. Theseantibiotics may be, but are not limited to, beta-lactams, macrolides,tetracyclines, quinolones, aminoglycosides, sulfonamides, glycopeptides,and oxazolidines. Moreover, the composition may be in a sterile form.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin. Such compositions contain a therapeuticallyeffective amount of the therapeutic composition, together with asuitable amount of carrier so as to provide the form for properadministration.

In a further embodiment, the compounds are in a pharmaceuticallyacceptable salt form or a form of free base. Examples ofpharmaceutically acceptable salts include, without limitation, thenontoxic inorganic and organic acid addition salts such as the acetate,aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate,embonate, enantate, formate, fumarate, glutamate, glycolate,hydrochloride, hydrobromide, lactate, maleate, alonate, mandelate,methanesulfonate, naphthalene-2-sulphonate, nitrate, perchlorate,phosphate, phthalate, salicylate, sorbate, stearate, succinate,sulphate, tartrate, toluene-p-sulphonate, and the like.

In one embodiment, the composition may further contain pharmaceuticallyacceptable ingredients including metal salts and buffers. Metal salts ofthe compounds include alkali metal salts, such as the sodium salt of acompound containing a carboxyl group. The composition may also containother acids such as oxalic acid, which may not be consideredpharmaceutically acceptable, but may be useful in the preparation ofsalts.

In one embodiment, the compounds may be provided in un-solvated orsolvated forms together with a pharmaceutically acceptable solvent(s)such as water, ethanol, and the like. Solvated forms may also includehydrated forms such as the monohydrate, the dihydrate, the hemihydrate,the trihydrate, the tetrahydrate, and the like. In general, solvatedforms are considered equivalent to un-solvated forms. In addition, thecompounds, their salts, and derivatives may be hydrated or anhydrous.

In one embodiment, the pharmaceutical composition comprising compoundsaccording to the invention, together with a conventional adjuvant,carrier, or diluent, may thus be placed into the form of solidsincluding tablets, filled capsules, powder and pellet forms, and liquidssuch as aqueous or non-aqueous solutions, suspensions, emulsions,elixirs, and capsules filled with the same. The composition may furthercomprise conventional ingredients in conventional proportions, with orwithout additional active compounds.

In a further embodiment, the composition is in the powder form. Thepharmaceutically accepted carrier is a finely divided solid which is ina mixture with the finely divided active compounds. In anotherembodiment, the composition is in the tablet form. The active componentis mixed with the pharmaceutically accepted carrier having the necessarybinding capacity in suitable proportions and compacted in desired shapeand size. Suitable carriers include magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like.

In a further embodiment, the composition is in other solid formsincluding capsules, pills, cachets, and lozenges which are suitable fororal administration.

In one embodiment, the current invention provides a pharmaceuticalcomposition for treating conditions involving bacterial infection,preferably Tuberculosis.

Methods for Bacterial Topoisomerase I Inhibition

In one embodiment, the current invention also provides methodscomprising the administration of an effective amount of thepharmaceutical composition for treating subjects infected with bacterialpathogens, and/or in need of inhibiting the activity of topoisomerase.The subjects may refer to any animal including, but not limited to,humans, non-human primates, rodents, and the like. In a preferredembodiment, the subject is a human. In another preferred embodiment, thehuman is infected with mycobacteria, in particular, M. tuberculosis, andNTM.

In one embodiment, the current invention provides methods for treating apatient with Tuberculosis, comprising the administration of thepharmaceutical composition. The composition described herein haseffective antibacterial activity and are selective for bacterialtopoisomerase I inhibition. In a further embodiment, the compositiontargets and inhibits MtbTopI.

Furthermore, it would be understood by those skilled in the art that themethods described in the present invention would not only apply totreatment in a subject, but could be applied to cell cultures, organs,tissues, or individual cells in vivo or in vitro, including tumors, andimmortalized cells isolated or derived from a subject.

The present invention also provides methods of inhibiting topoisomerasein bacteria, comprising the administration of an effective amount of oneor more of the compounds and/or the pharmaceutical compositions asdescribed herein to one or more bacteria. In one embodiment, thetopoisomerase is the type IA topoisomerase, preferably, bacterialtopoisomerase I, more preferably, EcTopI and MtbTopI.

Formulation and Administration

In one embodiment, the effective amount of said pharmaceuticalcomposition can be administered through oral, rectal, bronchial, nasal,topical, buccal, sub-lingual, transdermal, vaginal, intramuscular,intraperitoneal, intravenous, intra-arterial, intracerebral,interaocular administration or in a form suitable for administration byinhalation or insufflation, including powders and liquid aerosoladministration, or by sustained release systems such as semipermeablematrices of solid hydrophobic polymers containing the compound(s) of theinvention. Administration may be also by way of other carriers orvehicles such as patches, micelles, liposomes, vesicles, implants (e.g.microimplants), synthetic polymers, microspheres, nanoparticles, and thelike.

In specific embodiments, the compounds may be administered in the rangeof from 0.01 mg/kg body weight to 1 g/kg body weight, preferably, 1mg.kg to 500 mg/kg body weight, more preferably, 50 mg/kg to 500 mg/kgbody weight.

In one embodiment, the composition may be formulated for parenteraladministration e.g., by injection, for example, bolus injection orcontinuous infusion. In addition, the composition may be presented inunit dose form in ampoules, pre-filled syringes, and small volumeinfusion or in multi-dose containers with or without an addedpreservative. The compositions may be in forms of suspensions,solutions, or emulsions in oily or aqueous vehicles. The composition mayfurther contain formulation agents such as suspending, stabilizingand/or dispersing agents. In a further embodiment, the active ingredientof the composition according to the invention may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use.

In one embodiment, the composition may be formulated in aqueoussolutions for oral administration. The composition may be dissolved insuitable solutions with added suitable colorants, flavours, stabilisingand thickening agents, artificial and natural sweeteners, and the like.In addition, the composition may further be dissolved in solutioncontaining viscous material, such as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, or other well-knownsuspending agents.

In one embodiment, the composition is applied topically or systemicallyor via a combination of both. The composition may be formulated in theforms of lotion, cream, gel and the like.

In one embodiment, the composition can be applied directly to the nasalcavity by conventional means, for example with a dropper, pipette orspray. The compositions may be provided in single or multi-dose form.Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the active ingredient is provided in apressurized pack with a suitable propellant such as a chlorofluorocarbon(CFC) for example dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin.

Furthermore, the composition may be provided in the form of a drypowder, for example a powder mix of the compound in a suitable powderbase such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).Conveniently, the powder carrier will form a gel in the nasal cavity.The powder composition may be presented in unit dose form for example incapsules or cartridges of, e.g., gelatin, or blister packs from whichthe powder may be administered by means of an inhaler.

In one embodiment, the pharmaceutical composition is provided in unitdosage forms, wherein the composition in desired form is divided intounit doses containing appropriate quantities of the active component.The unit dosage form can be a packaged preparation, the packagecontaining discrete quantities such as packaged tablets, capsules, andpowders in vials or ampoules. Moreover, the unit dosage form can be acapsule, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form. In a preferred embodiment,tablet or capsule forms are for oral administration and liquid form arefor intravenous administration and continuous infusion.

The present invention also provides kits comprising the compounds and/orpharmaceutical compositions as described herein. The kits may further beused in the methods described herein. The kits may also include at leastone reagent and/or instruction for their use. Moreover, the kits mayinclude one or more containers filled with one or more compounds and/orpharmaceutical composition described in the present invention. The kitsmay also comprise a control composition, such as a control antibiotic.

The following are examples that illustrate the aforementionedembodiments and should not be construed as limiting.

EXAMPLES

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

Example 1 General Methods for Chemistry

All reagents and anhydrous solvents were purchased from Sigma-Aldrichand Fisher Scientific, and were used without further purification. Allreactions were monitored either by thin-layer chromatography (TLC) or byanalytical high performance liquid chromatography (HPLC) to detect thecompletion of reactions. TLC was performed using glass plates pre-coatedwith silica gel (0.25 mm, 60-Å pore size, 230-400 mesh, SorbentTechnologies, GA) impregnated with a fluorescent indicator (254 nm). TLCplates were visualized by exposure to ultraviolet light (UV).Hydrogenation reactions were performed employing domnick hunter NITROXUHP-60H hydrogen generator, USA. Microwave synthesis was performed usingBiotage Initiator 8 Exp Microwave System. Compounds were purified byflash column chromatography on silica gel using a Biotage Isolera Onesystem and a Biotage SNAP cartridge. ¹H and ¹³C NMR spectra wereobtained on a Bruker Avance DRX-400 instrument with chemical shifts (δ,ppm) determined using TMS as internal standard. Coupling constants (J)are in hertz (Hz). ESI mass spectra in either positive or negative modewere provided by Varian 500-MS IT Mass Spectrometer. High-resolutionmass spectra (HRMS) were obtained on an Agilent 6530 Accurate Mass Q-TOFLC/MS. The purity of compounds was determined by analytical HPLC using aGemini, 3 μm, C18, 110 Å column (50 mm×4.6 mm, Phenomenex) and a flowrate of 1.0 mL/min. Gradient conditions: solvent A (0.1% trifluoroaceticacid in water) and solvent B (acetonitrile): 0-2.00 min 100% A,2.00-7.00 min 0-100% B (linear gradient), 7.00-8.00 min 100% B, UVdetection at 254 and 220 nm.

6-Amino-2,3-difluorophenol (2)

2,3-Difluoro-6-nitrophenol (700 mg, 4 mmol) was dissolved in ethanol (5mL) and palladium on activated carbon (Pd/C) (84.8 mg, 20%) was added.The reaction was stirred at room temperature under H₂ atmosphere (1.0bar). After 7 h, all starting material was consumed and Pd/C wasfiltered through Celite. The solvent was evaporated under reducedpressure to afford 6-amino-2,3-difluorophenol (550.5 mg, 95% yield). ¹HNMR (400 MHz, CDCl₃): δ (ppm) 6.59 (dt, J=9.9, 8.6 Hz, 1H), 6.47-6.31(m, 1H), 4.27 (br s, 2H). ESI-HRMS: calc. for C₆H₆F₂NO [M+H]⁺: 146.0412,found: 146.0418.

Diethyl 2-(((3,4-difluoro-2-hydroxyphenyl)amino)methylene)malonate (3)

6-Amino-2,3-difluorophenol (2) (550 mg, 3.8 mmol) was dissolved inethanol (15 mL) and diethyl 2-(ethoxymethylene)malonate (819 mg, 3.8mmol) was added. The reaction was stirred at room temperature untilthere was no starting material left. Ethanol was removed and the residuewas purified by flash column chromatography on silica gel(EtOAc/hexane=1/3 to 3/1) to give product 3 as a brown solid (1.04 g,87%). ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 10.96 (d, J=14.0 Hz, 1H), 8.45(d, J=13.9 Hz, 1H), 7.31-7.22 (m, 1H), 6.98-6.86 (m, 1H), 4.20 (q, J=7.1Hz, 2H), 4.12 (q, J=7.1 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H), 1.24 (t, J=7.1Hz, 3H). ESI-MS: calc. for C₁₄H₁₄F₂NO₅ [M−H]⁻: 314.3, found: 314.3.

Ethyl 6, 7-difluoro-8-hydroxy-4-oxo-1, 4-dihydroquinoline-3-carboxylate(4)

Compound 3 (445 mg, 1.4 mmol) was added in a microwave sealed tube.Diphenyl ether (2.5 mL) was added and the tube was sealed with cap. Thereaction was then set up at 250 ° C. in a Biotage Microwave Initiatorinstrument for 30 min. Hexane was added and the solid was filtered andwashed with hexane. The product was then dried and obtained in 80% yield(299.4 mg), which was used for next step without further purification.¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.36 (s, 1H), 7.45 (dd, J=10.9, 7.9Hz, 1H), 4.21 (q, J=7.1 Hz, 2H), 1.27 (t, J=7.1 Hz, 3H). ESI-MS: calc.for C₁₂H₈F₂NO₄ [M−H]⁻: 268.2, found: 268.2.

Ethyl 5, 6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6a)

Compound 4 (269 mg, 1 mmol) and 1-chloro-2,4-dinitrobenzene (202 mg, 1mmol) were dissolved in DMF (2 mL). NaHCO₃ (252 mg, 3 mmol) was addedand the reaction was stirred at 100° C. until no starting materials weredetected by HPLC. The solid base was removed by filtration throughCelite. The filtrate was concentrated and the residue was purifiedthrough Biotage reverse phase C18 cartridge to give 220 mg of 6a asyellow solid (yield: 57%). ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.01 (s,1H), 8.15 (d, J=9.6 Hz, 1H), 8.05-8.01 (m, 2H), 7.60-7.54 (m, 1H), 4.27(q, J=7.2 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz, d₆-DMSO): δ(ppm) 170.4, 163.7, 145.8, 142.7, 138.1, 133.2, 129.4, 124.6, 122.2,120.8, 117.0, 113.0, 105.4, 105.2, 60.8, 14.2; ESI-MS: calc. forC₁₈H₁₀F₂N₂O₆Na [M+Na]⁺: 411.3, found: 411.2. ESI-HRMS: calc. forC₁₈H₁₁F₂N₂O₆ [M+H]⁺: 389.0580, found: 389.0582. HPLC purity: 100% (254nm), t_(R): 6.92 min; 100% (220 nm), t_(R): 6.92 min.

Compounds 6b-e were prepared according to the experimental procedureabove for the preparation of 6a.

Ethyl9-cyano-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate(6b)

Yellow solid. Yield: 59%. ¹NMR (400 MHz, d₆-DMSO): δ (ppm) 9.04 (s, 1H),8.11 (d, J=8.8 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.72 (dd, J=1.6 and 8.8Hz, 1H), 7.63-7.59 (m, 1H), 4.28 (q, J=7.2 Hz, 2H), 1.31 (t, J=7.2 Hz,3H); ESI-MS: calc. for C₁₉H₁₀F₂N₂O₄Na [M+Na]⁺: 391.3, found: 391.2.ESI-HRMS: calc. for C₁₉H₁₁F₂N₂O₄ [M+H]⁺: 369.0681, found: 369.0684. HPLCpurity: 100% (254 nm), t_(R): 6.73 min; 100% (220 nm), t_(R): 6.73 min.

Ethyl5,6-difluoro-3-oxo-9-(trifluoromethyl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate(6c)

Yellow solid. Yield: 10%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.05 (s,1H), 8.12 (d, J=8.8 Hz, 1H), 7.71 (d, J=1.6 Hz, 1H), 7.63-7.58 (m, 2H),4.27 (q, J=7.2 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H); ESI-MS: calc. forC₁₉H₁₀F₅NO₄Na [M+Na]⁺: 434.3, found: 434.1. ESI-HRMS: calc. forC₁₉H₁₁F₅NO₄ [M+H]⁺: 412.0603, found: 412.0603. HPLC purity: 100% (254nm), t_(R): 7.25 min; 100% (220 nm), t_(R): 7.25 min.

Ethyl9-acetyl-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate(6d)

Dark yellow solid. Yield: 38%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.97(s, 1H), 8.06-7.93 (m, 1H), 7.73 (d, J=8.4 Hz, 1H), 7.69 (s, 1H), 7.55(dd, J=10.2, 8.0 Hz, 1H), 4.27 (q, J=7.1 Hz, 2H), 2.58 (s, 3H), 1.33 (t,J=7.1 Hz, 3H). ESI-MS: calc. for C₂₀H₁₃F₂NNaO₅ [M+Na]³⁰ : 408.3, found:408.1. HPLC purity: 100% (254 nm), t_(R): 6.62 min; 100% (220 nm),t_(R): 6.62 min.

Ethyl10-chloro-5,6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate(6e)

Dark yellow solid. Yield: 40%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.13(s, 1H), 8.48 (s, 1H), 8.12 (s, 1H), 7.69-7.60 (m, 1H), 4.30 (q, J=7.0Hz, 2H), 1.33 (t, J=7.1 Hz, 3H). ESI-MS: calc. for C₁₈H₉ClF₂N₂NaO₆[M+Na]⁺: 445.0, found: 445.1. HPLC purity: 98.5% (254 nm), t_(R): 6.92min; 96.9% (220 nm), t_(R): 6.92 min.

5,6-Difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (7a)

Compound 6a (180 mg, 0.46 mmol) was dissolved in AcOH (15 mL).Hydrochloric acid (1 N, 2 mL) was added and the reaction was stirredunder reflux for 2 h. Upon completion, water was added and yellow solidwas precipitated. The solid was collected by filtration and washed withwater, then dried and afforded product 7a (150 mg) as yellow solid in91% yield. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.36 (s, 1H), 8.42 (d,J=9.2 Hz, 1H), 8.17 (d, J=2.8 Hz, 1H), 8.09 (dd, J=2.4 and 9.2 Hz, 1H),7.86-7.82 (m, 1H); ESI-MS: calc. for C₁₆H₇F₂N₂O₆ [M+H]⁺: 361.2, found:361.3. ESI-HRMS: calc. for C₁₆H₇F₂N₂O₆ [M+H]⁺: 361.0267, found:361.0268. HPLC purity: 100% (254 nm), t_(R): 6.79 min; 100% (220 nm),t_(R): 6.79 min.

Compounds 7b-e were prepared according to the experimental procedureabove for the preparation of 7a.

9-Cyano-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (7b)

Yellow solid. Yield: 36%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.34 (s,1H), 8.35 (d, J=8.8 Hz, 1H), 7.98 (d, J=1.6 Hz, 1H), 7.82 (d, J=2.4 Hz,1H), 7.78-7.75 (m, 2H); ESI-MS: calc. for C₁₇H₇F₂N₂O₄ [M+H]⁺: 341.2,found: 341.1. ESI-HRMS: calc. for C₁₇H₇F₂N₂O₄ [M+H]⁺: 341.0368, found:341.0378. HPLC purity: 95.1% (254 nm), t_(R): 6.37 min; 97.7% (220 nm),t_(R): 6.37 min.

5,6-Difluoro-3-oxo-9-(trifluoromethyl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (7c)

Yellow solid. Yield: 95%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.35 (s,1H), 8.37 (d, J=8.4 Hz, 1H), 7.84-7.79 (m, 2H), 7.65-7.62 (dd, J=1.2 and8.8 Hz, 1H); ESI-MS: calc. for C₁₇H₇F₅NO₄ [M+H]⁺: 384.2, found: 384.1.ESI-HRMS: calc. for C₁₇H₇F₅NO₄ [M+H]⁺: 384.0290, found: 384,0293. HPLCpurity: 100% (254 nm), t_(R): 7.15 min; 100% (220 nm), t_(R): 7.15 min.

9-Acetyl-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (7d)

Yellow solid. Yield: 62%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.36 (s,1H), 8.30 (d, J=11.2 Hz, 1H), 7.85-7.80 (m, 3H), 2.63 (s, 3H); ESI-MS:calc. for C₁₈H₁₀F₂NO₅ [M+H]⁺: 358.3, found: 358.2. ESI-HRMS: calc. forC₁₈H₁₀F₂NO₅ [M+H]⁺: 358.0522, found: 358.0521. HPLC purity: 97.3% (254nm), t_(R): 6.70 min; 97.2% (220 nm), t_(R): 6.70 min.

10-Chloro-5,6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (7e)

Brown solid. Yield: 39%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.43 (s,1H), 8.74 (s, 1H), 8.18 (s, 1H), 7.83-7.79 (m, 1H); ESI-MS: calc. forC₁₆H₆ClF₂N₂O₆ [M+H]⁺: 394.7, found: 394.9. ESI-HRMS: calc. forC₁₆H₆F₂N₂ClO₆ [M+H]⁺: 394.9877, found: 394.9879. HPLC purity: 95.4% (254nm), t_(R): 6.90 min; 97.0% (220 nm), t_(R): 6.90 min.

9-Amino-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (1)

Compound 7a (150 mg, 0.42 mmol) was added to a mixture of AcOH/HCl(1/1). SnCl₂ (236 mg, 1.25 mmol) was added and the reaction was stirredunder reflux for 2 h. No starting material was observed in the reactionand then water was added. The large amount of solid precipitated and wascollected by filtration. Product 1 was obtained in 86% yield (236 mg) asyellow solid. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.08 (s, 1H), 7.79 (d,J=9.2 Hz, 1H), 7.76-7.73 (m, 1H), 6.48 (dd, J=2.4 and 9.2 Hz, 1H), 6.43(d, J=2.4 Hz, 1H); ESI-MS: calc. for C₁₆H₉F₂N₂O₄ [M+H]⁺: 331.3, found:331.1. ESI-HRMS: calc. for C₁₆H₉F₂N₂O₄ [M+H]⁺: 331.0525, found:331.0526. HPLC purity: 100% (254 nm), t_(R): 6.47 min; 100% (220 nm),t_(R): 6.47 min.

Ethyl-5-fluoro-6-(4-methylpiperazin-1-yl)-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate(9)

Compound 6a (159 mg, 0.4 mmol) was dissolved in pyridine (1 mL) and1-methylpiperazine (120 mg, 1.2 mmol) was added. The reaction was heatedto 110° C. until no starting material was observed. Pyridine was removedunder reduced pressure and the residue was purified by flash columnchromatography on silica gel (MeOH/CH₂Cl₂=1/19), affording product 9(100.1 mg) in 53% yield as orange solid. ¹H NMR (400 MHz, d₆-DMSO): δ(ppm) 8.87 (d, J=1.6 Hz, 1H), 8.04-7.97 (m, 2H), 7.87 (t, J=1.2 Hz, 1H),7.32 (dd, J=1.2 and 8.0 Hz, 2H), 4.25 (q, J=7.2 Hz, 2H), 3.34 (s, 4H),3.27 (s, 4H), 2.27 (s, 3H), 1.32 (t, J=7.2 Hz, 3H); ESI-MS: calc. forC₂₃H₂₂FN₄O₆ [M+H]⁺: 469.4, found: 469.3. HPLC purity: 100% (254 nm),t_(R): 5.48 min; 100% (220 nm), t_(R): 5.48 min.

5-Fluoro-6-(4-methylpiperazin-1-yl)-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (10a)

Compound 7a (108 mg, 0.3 mmol) was dissolved in pyridine (4 mL), andthen the reaction was heated to 90° C. 1-Methylpiperazine (100 μL, 0.9mmol) was added and the reaction was stirred under nitrogen atmosphereuntil there was no starting material. Upon completion, pyridine wasremoved under reduced pressure and the residue was dissolved in ethanol(10 mL) and heated under reflux for additional 30 min. The solid wasfiltered and washed with water, then dried to obtain product 10a (60 mg)in 45% yield as yellow solid. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.18(s, 1H), 8.29 (d, J=8.0 Hz, 1H), 8.02-7.99 (m, 2H), 7.53 (d, J=12.6 Hz,1H), 3.30 (br s, overlapping with H₂O peak, 4H), 2.57 (br s, 4H), 2.32(s, 3H); ESI-MS: calc. for C₂₁H₁₈FN₄O₆ [M+H]⁺: 441.4, found: 441.4.ESI-MS: calc. for C₂₁H₁₈FN₄O₆ [M+H]⁺: 441.1205, found: 441.1216. HPLCpurity: 100% (254 nm), t_(R): 5.37 min; 100% (220 nm), t_(R): 5.37 min.

Compounds 10b and 11a-k were prepared following the similar procedurefor the preparation of 10a.

5-Fluoro-9-nitro-3-oxo-6-(piperazin-1-yl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (10b)

Yellow solid. Yield: 84%, ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.94 (s,1H), 7.66 (d, J=8.8 Hz, 1H), 7.45 (d, J=12.0 Hz, 1H), 6.43-6.36 (m, 2H),3.22 (s, 4H), 2.87 (s, 4H); ESI-MS: calc. for C₂₀H₁₆FN₄O₆ [M+H]⁺: 427.4,found: 427.2. ESI-HRMS: calc. for C₂₀H₁₆FN₄O₆ [M+H]⁺: 427.1048, found:427.1041. HPLC purity: 100% (254 nm), t_(R): 5.36 min; 100% (220 nm),t_(R): 5.36 min.

9-Amino-5-fluoro-6-(4-methylpiperazin-1-yl)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11a)

Yellow solid. Yield: 48%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.94 (s,1H), 7.65 (d, J=9.2 Hz, 1H), 7.45 (d, J=12.0 Hz, 1H), 6.43-6.36 (m, 2H),5.77 (s, 2H), 3.31 (s, 4H), 2.57 (s, 4H), 2.32 (s, 3H); ESI-MS: calc.for C₂₁H₂₀FN₄O₄ [M+H]⁺: 411.4, found: 411.2. ESI-MS: calc. forC₂₁H₂₀FN₄O₄ [M+H]⁺: 411.1463, found: 411.1466. HPLC purity: 100% (254nm), t_(R): 5.22 min; 100% (220 nm), t_(R): 5.22 min.

9-Amino-5-fluoro-3-oxo-6-(piperazin-1-yl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11b)

Yellow solid. Yield: 97%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.97 (s,1H), 7.68 (d, J=9.2 Hz, 1H), 7.48 (d, J=12.4 Hz, 1H), 6.44-6.38 (m, 2H),5.77 (s, 2H), 3.22 (s, 4H), 2.86 (s, 4H); ESI-MS: calc. for C₂₀H₁₈FN₄O₄[M+H]⁺: 397.4, found: 397.2. ESI-HRMS: calc. for C₂₀H₁₈FN₄O₄ [M+H]⁺:397.1307, found: 397.1303. HPLC purity: 100% (254 nm), t_(R): 5.20 min;100% (220 nm), t_(R): 5.20 min.

9-Amino-5-fluoro-6-(4-methylpiperidin-1-yl)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11c)

Yellow solid. Yield: 33%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.92 (s,1H), 7.63 (d, J=8.0 Hz, 1H), 7.43 (d, J=11.6 Hz, 1H), 6.42-6.36 (m, 2H),5.75 (s, 2H), 3.13 (t, J=10.8 Hz, 2H), 1.71 (d, J=11.2 Hz, 2H), 1.57 (s,1H), 1.32-1.26 (m, 2H), 0.98 (s, 3H); ¹³C NMR (100 MHz, d₆-DMSO): δ(ppm) 175.4, 166.4, 150.7, 144.6, 138.5, 136.0, 131.7, 125.3, 119.6,119.5, 117.6, 112.2, 111.1, 107.0, 104.0, 101.2, 51.1, 35.1, 30.6, 22.5;ESI-MS: calc. for C₂₂H₂₁FN₃O₄ [M+H]⁺: 410.4 found: 410.2. ESI-HRMS:calc. for C₂₂H₂₁FN₃O₄ [M+H]⁺: 410.1511, found: 410.1510. HPLC purity:100% (254 nm), t_(R): 6.38 min; 100% (220 nm), t_(R): 6.38 min.

9-Amino-5-fluoro-6-morpholino-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11d)

Yellow solid. Yield: 71%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.93 (s,1H), 7.64 (d, J=8.8 Hz, 1H), 7.45 (d, J=12.0 Hz, 1H), 6.43-6.37 (m, 2H),5.75 (s, 2H), 3.69 (s, 4H), 3.28 (s, 4H); ESI-MS: calc. for C₂₀H₁₇FN₃O₅[M+H]⁺: 398.4, found: 398.2. ESI-HRMS: calc. for C₂₀H₁₇FN₃O₅ [M+H]⁺:398.1147, found: 398.1146. HPLC purity: 100% (254 nm), t_(R): 6.38 min;100% (220 nm), t_(R): 6.38 min.

9-Amino-5-fluoro-3-oxo-6-(phenethylamino)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11e)

Yellow solid. Yield: 55%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.90 (s,1H), 7.61 (d, J=9.2 Hz, 1H), 7.45 (d, J=13.2 Hz, 1H), 7.29-7.18 (m, 5H),6.43-6.37 (m, 2H), 6.15 (br s, 1H), 5.76 (br s, 2H), 3.68-3.61 (m, 2H),2.89-2.85 (m, 2H); ESI-MS: calc. for C₂₄H₁₈FN₃O₄ [M+H]⁺: 432.4, found:432.1. ESI-HRMS: calc. for C₂₄H₁₉FN₃O₄ [M+H]⁺: 432.1354, found:432.1362. HPLC purity: 100% (254 nm), t_(R): 7.07 min; 100% (220 nm),t_(R): 7.07 min.

6-(((3s,5s,7s)-Adamantan-1-yl)amino)-9-amino-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11f)

Brown solid. Yield: 29%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 9.00 (s,1H), 7.71 (d, J=9.1 Hz, 1H), 7.54 (d, J=10.7 Hz, 1H), 6.50-6.38 (m, 2H),5.81 (s, 2H), 4.34 (s, 1H), 2.09-2.04 (m, 3H), 1.85 (s, 6H), 1.65-1.55(m, 6H). ESI-MS: calc. for C₂₆H₂₃FN₃O₄ [M−H]⁺: 460.2, found: 460.0. HPLCpurity: 85.2% (254 nm), t_(R): 7.41 min; 91.1% (220 nm), t_(R): 7.41min.

6-([1,4′-Bipiperidin]-1′-yl)-9-amino-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11g)

Yellow solid. Yield: 64%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.98 (s,1H), 7.68 (d, J=4.0 Hz, 1H), 7.49 (d, J=9.6 Hz, 1H), 6.45-6.41 (m, 2H),5.77 (br s, 2H), 3.20-3.15 (m, 1H), 1.84-1.82 (m, 3H), 1.64-1.41 (m,14H), 1.06-1.04 (m, 1H); ESI-MS: calc. for C₂₆H₂₈FN₄O₄ [M+H]⁺: 479.5,found: 479.3. ESI-HRMS: calc. for C₂₆H₂₈FN₄O₄ [M+H]⁺: 479.2089, found:479.2093. HPLC purity: 100% (254 nm), t_(R): 5.53 min; 100% (220 nm),t_(R): 5.53 min.

9-Amino-5-fluoro-6-((2-morpholinoethyl)amino)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11h)

Yellow solid. Yield: 74%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.90 (s,1H), 8.55 (s, 1H), 7.46 (d, J=6.0 Hz, 1H), 7.39-7.34 (m, 2H), 6.42-6.37(m, 2H), 5.95 (s, 1H), 5.78 (br s, 2H), 2.57 (s, 2H), 2.44 (br s, 6H);ESI-MS: calc. for C₂₂H₂₂FN₄O₅ [M+H]⁺: 441.4, found: 441.3. ESI-MS: calc.for C₂₂H₂₂FN₄O₅ [M+H]⁺: 441.1569, found: 441.1579. HPLC purity: 97.2%(254 nm), t_(R): 5.22 min; 99.3% (220 nm), t_(R): 5.22 min.

9-Amino-6-(cyclohexylamino)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11i)

Brown solid. Yield: 74%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.86 (s,1H), 7.58-7.56 (m, 1H), 7.43-7.40 (m, 1H), 6.40 (s, 1H), 5.72 (s, 2H),5.40-5.38 (m, 1H), 1.92 (s, 3H), 1.73-1.67 (m, 3H), 1.31-1.22 (m, 6H);ESI-MS: calc. for C₂₂H₂₁FN₃O₄ [M+H]⁺: 410.4, found: 410.3. ESI-HRMS:calc. for C₂₂H₂₁FN₃O₄ [M+H]⁺: 410.1511, found: 410.1494. HPLC purity:100% (254 nm), t_(R): 7.27 min; 100% (220 nm), t_(R): 7.27 min.

9-Amino-6-(cyclopentylamino)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11j)

Black solid. Yield: 63%. H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.91 (s, 1H),7.62 (d, J=8.8 Hz, 1H), 7.47 (d, J=12.4 Hz, 1H), 6.46-6.41 (m, 2H), 5.73(s, 2H), 5.53 (d, J=2.4 Hz, 1H), 4.30 (s, 1H), 1.93 (br s, 4H), 1.73 (brs, 4H); ESI-MS: calc. for C₂₁H₁₉FN₃O₄ [M+H]⁺: 396.4, found: 396.1.ESI-HRMS: calc. for C₂₁H₁₉FN₃O₄ [M+H]⁺: 396.1354, found: 396.1358. HPLCpurity: 96.7% (254 nm), t_(R): 7.07 min; 96.0% (220 nm), t_(R): 7.06min.

9-Amino-5-fluoro-6-(hexylamino)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (11k)

Yellow solid. Yield: 49%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.88 (s,1H), 7.64 (s, 1H), 7.44 (s, 1H), 6.56-6.36 (m, 2H), 5.88-5.50 (m, 2H),1.57 (s, 2H), 1.27 (s, 7H), 0.84 (s, 4H); ESI-MS: calc. for C₂₂H₂₃FN₃O₄[M+H]⁺: 412.4, found: 412.2. ESI-HRMS: calc. for C₂₂H₂₃FN₃O₄ [M+H]⁺:412.1667, found: 412.1672. HPLC purity: 100% (254 nm), t_(R): 7.47 min;100% (220 nm), t_(R): 7.47 min.

9-acetamido-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (12)

To the solution of 1 (33 mg, 0.1 mmol) and pyridine (1 mL), aceticanhydride (12.2 mg, 0.12 mmol) was added. The reaction was stirred at80° C. for 3 h, then at 100° C. until no starting material was detectedby HPLC. The solid was filtered and dried to give 34.1 mg of 12 in 92%yield. Yellow solid. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 10.31 (s, 1H),9.20 (s, 1H), 8.05 (d, J=9.2 Hz, 1H), 7.78 (t, J=8.0 Hz, 1H), 7.66 (s,1H), 7.29 (d, J=7.6 Hz, 1H), 2.06 (s, 3H); ESI-MS: calc. forC₁₈H₁₁F₂N₂O₅ [M+H]⁺: 373.3, found: 373.2. ESI-MS: calc. for C₁₈H₁₁F₂N₂O₅[M+H]⁺: 373.0631, found: 373.0638. HPLC purity: 98.6% (254 nm), t_(R):6.42 min; 99.1% (220 nm), t_(R): 6.42 min.

Compounds 14 and 15a-b were prepared at 70° C. following the similarprocedure for the preparation of 10a

(S)-9-amino-6-(3-((tert-butoxycarbonyl)amino)pyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (15a)

Yellow solid. Yield: 86%. ¹H NMR (400 MHz, d₆-DMSO): δ (ppm) 8.77 (s,1H), 7.49 (d, J=9.6 Hz, 1H), 7.31 (d, J=14.0 Hz, 1H), 7.17 (d, J=5.6 Hz,1H), 6.38 (dd, J=2.4 and 9.2 Hz, 2H), 6.29 (d, J=2.0 Hz, 1H), 5.67 (s,2H), 3.82-3.80 (m, 5H), 2.05-2.04 (m, 1H), 1.83 (br s, 1H), 1.38 (s,9H); ESI-MS: calc. for C₂₅H₂₆FN₄O₆ [M+H]⁺: 497.5, found: 497.3.ESI-HRMS: calc. for C₂₅H₂₆FN₄O₆ [M+H]⁺: 497.1831, found: 497.1831. HPLCpurity: 100% (254 nm), t_(R): 6.94 min; 100% (220 nm), t_(R): 6.94 min.

(S)-6-(3-aminopyrrolidin-1-yl)-5-fluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (16)

Compound 16 was prepared following the same procedure for the synthesisof 7. Brown solid. Yield: 32% (two steps). ¹H NMR (400 MHz, d₆-DMSO): δ(ppm) 9.10 (s, 1H), 8.41 (s, 2H), 8.25 (d, J=9.3 Hz, 1H), 8.09 (s, 1H),8.01 (d, J=7.2 Hz, 1H), 7.48 (d, J=13.6 Hz, 1H), 4.07-3.74 (m, 5H), 2.28(s, 1H), 2.08 (s, 1H). ESI-MS: calc. for C₂₀H₁₆FN₄O₆ [M+H]⁺: 427.4,found: 427.1. HPLC purity: 100% (254 nm), t_(R): 5.45 min; 100% (220nm), t_(R): 5.46 min.

(S)-9-amino-6-(3-aminopyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (17a)

Compound 15a (85 mg, 0.17 mmol) was dissolved in 10 mL of hydrochloricacid (1 N). The reaction was stirred under reflux for 2.5 h. The solventwas removed and ethanol (10 mL) was added. The mixture was heated underreflux for 30 min. The solid was collected by filtration and dried toyield 52 mg of 17a in 78% yield. Yellow solid. ¹H NMR (400 MHz,d₆-DMSO): δ (ppm) 8.94 (s, 1H), 8.24 (s, 3H), 7.65 (d, J=9.2 Hz, 1H),7.50 (d, J=13.6 Hz, 1H), 6.46-6.40 (m, 2H), 3.99-3.85 (m, 5H), 2.34-2.26(m, 1H), 2.09-1.98 (m, 1H); ESI-MS: calc. for C₂₀H₁₈FN₄O₄ [M+H]⁺: 397.4,found: 397.1. ESI-MS: calc. for C₂₀H₁₈FN₄O₄ [M+H]⁺: 397.1307, found:397.1298. HPLC purity: 100% (254 nm), t_(R): 5.20 min; 100% (220 nm),t_(R): 5.20 min.

(R)-9-amino-6-(3-aminopyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (17b)

Compound 17b was prepared using the same procedure for the synthesis of17a. Brown solid. Yield: 48% (two steps). ¹H NMR (400 MHz, d₆-DMSO): δ(ppm) 8.88 (s, 1H), 8.31 (s, 3H), 7.59 (d, J=9.2 Hz, 1H), 7.43 (d,J=13.2 Hz, 1H), 6.42(d, J=9.2 Hz, 1H), 6.38 (s, 1H), 3.98-3.88 (m, 2H),3.73-3.68 (m, 3H), 2.30-2.25 (m, 1H), 2.06-2.03 (m, 1H); ESI-MS: calc.for C₂₀H₁₈FN₄O₄ [M+H]⁺: 397.4, found: 397.2. ESI-HRMS: calc. forC₂₀H₁₈FN₄O₄ [M+H]⁺: 397.1307, found: 397.1302. HPLC purity: 100% (254nm), t_(R): 5.24 min; 100% (220 nm), t_(R): 5.24 min.

Example 2 Validation of the Chemistry

9-amino-5, 6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid (1) was synthesized and its biochemical activity tested against E.coli topoisomerase I. Resynthesized fluoroquinophenoxazine hit 1 showedreproducible topoisomerase I inhibitory activity with an IC₅₀ of 1.95μM. The synthesis of 1 is shown in Scheme 1 (FIG. 2).

Briefly, commercially available 2,3-difluoro-6-nitrophenol washydrogenated using Pd/C (20 mol %) as the catalyst to the correspondingamino product 2, which can be used in next step without furtherpurification. Subsequent reaction of the aniline derivative 2 withdiethyl 2-(ethoxymethylene)malonate under ambient temperature gave 3 in87% yield. For next nucleophilic displacement cyclization, an improvedprotocol was developed for this intramolecular cyclization reactionunder microwave irradiation at 250° C. instead of conventional heating[30].

Following simple filtration, 4 was obtained in 80% yield. Subsequently,4 reacted with 1-chloro-2,4-dinitrobenzene (5a) in DMF at 100° C. togive the desired tetracyclic product 6a in 57% yield. Upon treatment of6a in acidic condition (AcOH/HCl) under reflux for 4 h, the freecarboxylic acid derivative 7a was obtained by simple filtration in 91%yield. Finally, hydrogenation of 7a under H₂ (1.0 bar) using FeSO₄.6H₂Oas the catalyst failed to yield the amine product 1 after 14 h. However,when SnCl₂ was used as catalyst and AcOH as the solvent, the nitro groupwas smoothly reduced into the amino group under reflux for 3 h and thefinal product 1 was obtained in 86% yield.

Under the optimized conditions, the structural diversity of 1 wasexpended to evaluate the effect of various substituents on thequinophenoxazine skeleton and to explore their structure-activityrelationship (SAR). First, we used various substrates 5b-e in Scheme 2in an effort to generate a focused set of quinophenoxazine derivatives7b-e. It is worthwhile noting that a dramatic difference was observed interms of the reactivity of the substrate 5 with different electronicand/or physiochemical properties. For example, 5b bearing anelectron-withdrawing nitrile group facilitated the completion ofcyclization in 2 h and 6b was obtained in 59% yield. On the other hand,5c with the lipophilic trifluoromethyl group could finish the reactionby extending the reaction time, affording the desired product 6c in alower yield (10%). In addition, when the substrate 5d with an acetylgroup was tried, 6d was obtained in 38% yield. However, for thesubstrate with the corresponding fluorine substitution, no reactionoccurred even when the reaction temperature was raised to 120° C.Furthermore, more harsh conditions were tried in an attempt tofacilitate the reaction by heating the reaction to 160° C. in a sealedpressure tube or heating the reaction to 200° C. under microwaveirradiation, but with not much success. In the case of 5e withadditional nitro and chlorine substituents, the reaction proceededsmoothly and 6e was obtained in 40% yield. Final ester hydrolysis wasperformed in acetic acid/hydrochloric acid under reflux, affording freecarboxylic acid products 7b-e in 36-95% yields.

In the course of antibacterial evaluation, compound 1 lost 2-4 foldwhole cell antibacterial activity after 4 weeks of storage, indicatingthat 1 may have stability and/or solubility issues. From ¹H NMRexperiments, it was noted that 1 could be easily precipitated out ind₆-DMSO solvent and no degradation-related evidence was observedfollowing several weeks of monitoring 1 in both d₆-DMSO NMR and HPLCexperiments (data not shown). Thus, to enhance the overall solubilityprofile of this class of quinophenoxazine derivatives, a variety ofsolubilizing and polar groups were introduced into thefluoroquinophenoxazine scaffold by displacing the 6-fluorine atom of 6a,7a, or 1 with different amine functionalities such as piperazine,1-methylpiperazine, and morpholine (Scheme 3).

Specifically, 6a reacted with 1-methylpiperazine in pyridine at 110° C.to give 9 in 53% yield. Accordingly, 7a reacted with 1-methylpiperazineand piperazine in pyridine under nitrogen atmosphere at 90° C., and bothreactions proceeded smoothly to afford 10a and 10b in 45% and 84%yields, respectively [31].

In the cases of 1 and some other amine substrates, notably, reactiontemperature appeared to play a critical role in this nucleophilicdisplacement reaction. For example, when morpholine was used as anucleophile, the reaction became complicated under 90° C., which issuitable for 1-methylpiperazine and piperazine. In contrast, whentemperature was reduced to 70° C., the reaction could be completed in 16h to produce 11d in 71% yield. Other functional amines were alsosubjected to this substitution reaction, and most of the reactions couldlead to the desired products 11c-k under 90° C. in moderate yieldsexcept for 1-adamantylamine.

The reaction of 1 with 1-adamantylamine could finish under reflux after4 days in 29% yield, presumably due to steric effect. In addition, toinvestigate the effect of the free amine functionality of 1 ontopoisomerase inhibition and antibacterial activity, we next tried toprotect the free amino group with acetyl functionality. The N-acetylderivative 12 was synthesized from 1 and acetic anhydride in pyridine at80-100° C. and the solid product was collected by simple filtration inhigh yield (92%).

Finally, to evaluate the potential stereospecific effect at the 6position of fluoroquinophenoxazine derivatives on biological activity,we designed and synthesized several chiral fluoroquinophenoxazine aminederivatives from 6a or 1 and chiral amine building blocks [31, 32]. Thenucleophilic substitution reaction of 1 and (S)-3-(Boc-amino)pyrrolidine(13a) in pyridine was completed in 20 h, affording 15a in 86% yield.Subsequent N-Boc deprotection of 15a produced 17a in 78% yield upon thetreatment with diluted hydrochloric acid. With regard to the reaction of1 and the corresponding (R)-3-(Boc-amino)pyrrolidine (13b), thesubstituted compound 15b could not be obtained by filtration upon thecompletion of reaction. Therefore, the crude product 15b was used forthe following deprotection reaction and the correspondingfluorophenoxazine derivative 17b (R) was obtained in 48% yield over twosteps. Accordingly, compound 16 was synthesized from 6a as the startingmaterial in 32% yield over two steps (Scheme 4).

Example 3 Inhibition of E. coil Topoisomerase I Relaxation Activity

Recombinant E. coli topoisomerase I and gyrase expressed in E. coli werepurified as described previously [36, 37].

All the synthesized target molecules were tested for the ability toinhibit the relaxation activity of E. coli topoisomerase I intarget-based assay, as well as against a panel of bacterial strainsincluding the wild-type E. coli MG1655 K12 strain, E. coli strainBAS3023 with imp mutation conferring membrane permeability to smallmolecules [38, 39], the wild-type Gram-positive B. subtilis (ATCC 6633)strain, and M. tuberculosis (H₃₇Rv). The results are summarized in Table1.

E. coli Topoisomerase I Relaxation Activity Inhibition Assay

The relaxation activity of E. coli topoisomerase I was assayed in abuffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin,and 0.5 mM MgCl₂. Half microliter from the appropriate stock solutionsof compounds dissolved in the solvent (DMSO) or the solvent alone(control) was mixed with 9.5 μL of the reaction buffer containing 10 ngof enzyme before the addition of 10 μL of reaction buffer containing 200ng of supercoiled pBAD/Thio plasmid DNA purified by cesium chloridegradient as substrate. Following incubation at 37° C. for 30 min, thereactions were terminated by the addition of 4 μL of a stop buffer (50%glycerol, 50 mM EDTA, and 0.5% (v/v) bromophenol blue), and analyzed byagarose gel electrophoresis. The gels were stained in ethidium bromideand photographed under UV light.

E. coli Topoisomerase I Inhibition

Biochemical evaluation for inhibition of the relaxation activity of E.coli topoisomerase I revealed that the majority of our synthesizedcompounds possessed good activity against E. coli topoisomerase I. Onthe basis of these topoisomerase I inhibition data (Table 1), The 9position substituent plays a very important role in topoisomerase Iinhibitory activity. Among the 5,6-difluoroquinophenoxazine derivatives,the hit compound 1 with the electron-donating 9-NH₂ group showed themost potent activity (IC₅₀=1.95 μM) against E. coli topoisomerase I.Both free carboxylic acid 7c and its ethyl ester derivative 6c with the9-CF₃ functionality were inactive against topoisomerase I when tested at125 μM. In addition, compared to 1 (9-NH₂, 1.95 μM), all the other5,6-difluoro derivatives with 9-substituted electron-withdrawing groups(7a with 9-NO₂, 15.6 μM; 7b with 9-CN, 31.25 μM; 7d with 9-Ac, 15.6 μM;7e with 9-NO₂ and 10-Cl, 31.25 μM) were 8-16 fold less active with IC₅₀values ranging from 15.6-31.25 μM. Thus, the topoisomerase I inhibitoryactivity among these derivative is 1>7a, 7d>7b, 7e.

In general, the basic amine functionality at the 6 positionsignificantly enhanced topoisomerase I inhibitory activity. For example,the 6-substituted amine derivatives 11a with 6-methylpiperazinyl, 11bwith piperazinyl, 11d with morpholino, 11g with bipiperidinyl, 11h withmorpholinoethyl, as well as the 6-substituted aminopyrrolidinylderivatives 16, 17a, and 17b demonstrated the most potent topoisomeraseI inhibitory activity with IC₅₀ values of 0.24-0.97 μM; and within thisgroup, 11d and 11h with the morpholino group had an IC₅₀ value of 0.97μM. In contrast, all the other 6-substituted amine derivatives with amore lipophilic side chain, including 11c with methylpiperidinyl, 11ewith phenethyl, 11f with adamantanyl, 11i with cyclohexyl, 11j withcyclopentyl, 11k with n-hexyl, and 15a with t-Boc-aminopyrrolidinyl,showed weaker topoisomerase I inhibition with IC₅₀ values ranging from3.9 to 15.6 μM. Notably, both 6-substituted aminopyrrolidinyl S- andR-stereoisomers 17a and 17b exhibited the same topoisomerase Iinhibitory activity (IC₅₀=0.48-0.97 μM), suggesting the stereochemistryat the 6 position is not required for topoisomerase I inhibition. iii)Esterification of the carboxylic acid group had little effect on the E.coli topoisomerase I inhibitory activity by comparing 6a and 7a(IC₅₀=15.6 μM), 6b and 7b (IC₅₀=31.25 μM), as well as 6c and 7c(IC₅₀>125 μM), indicating the ethyl ester functionality is tolerated fortopoisomerase I enzyme inhibition. Representative inhibition results of11a and 11b against E. coli topoisomerase relaxation activity are shownin FIG. 6.

TABLE 1 E. coli topoisomerase I inhibition and whole cell antibacterialactivities (μM) of fluoroquinophenoxazine derivatives^(a) Topoisomeraseinhibitory activity (IC₅₀, μM) Whole cell based antibacterial activity(MIC, μM) E. coli E. coli Human Human E. coli E. coli B. subtilis M.Vero topo I DNA gyrase topo I topo IIα Imp4213 (MG1655) (ATCC 6633)tuberculosis cell Compd (type IA) (type IIA) (type IB) (type IIA)(BAS3023) WT WT (H₃₇Rv) IC₅₀ SI^(b)  1 1.95 >125 31.3 >5000.78-1.56 >200 6.25 11.2 75.7 6.8  6a 15.6 50 >200 25  6b31.25 >200 >200 200  6c >125 >200 >200 >200  7a 15.6 0.78 200 12.5  7b31.25 200 >200 >200  7c >125 >200 >200 25  7d 15.6 >200 >200 >200  7e31.25 62.5 31.25 125 0.39 50 3.12 29 >127 >4.4  9 1.95 25 >200 50 10a1.95 62.5-125   31.25 250-500 1.56 100 0.78 19 95 5.0 10b3.9 >200 >200 >200 11a 0.48 15.6-31.25 15.6 3.9-7.8 0.78 6.25 0.78 7.629 3.8 11b 0.24 7.8-15.6 7.8 1.95-3.9  0.39 >200 25 29.5 >126 >4.3 11c3.9 3.12 >200 0.78 11d 0.97 7.8 15.6 15.6 0.19-0.39 >200 0.19 3.5 24.77.1 11e 3.9 50 >200 25-50 21.6 11f 3.9-7.8 3.12 >200 12.5 38.4 30.7 0.811g 0.48 15.6 3.9 1.95-3.9  0.39-0.78 >200 1.56 2.5 24.4 9.8 11h 0.9715.6-31.25 7.8-15.6 3.9-7.8 0.78 >200 1.56 21.6 43.0 2.0 11i 3.9 25 >20012.5 11j 3.9 25 >200 12.5 11k 15.6 >200 >200 >200 12 7.8 >200 >200 >20015a 3.9 12.5 >200 12.5 16 0.48 3.9 7.8 1.95-3.9  1.56 >200 0.78 >50 >5017a 0.48-0.97 3.9 3.9 0.97-1.95 1.56 >200 6.25 >63.1 >63.1 17b 0.48-0.973.9 3.9 3.9 6.25 >200 12.5 >63.1 >63.1 ^(a)Blank cells indicate NotDetermined. ^(b)Selectivity index = cytotoxic IC₅₀ against Verocells/MIC against M. tuberculosis.

Example 4 Selectivity and Specificity Against Other DNA TopoisomeraseEnzymes DNA Gyrase Supercoiling Inhibition Assay

DNA gyrase supercoiling assays were carried out by mixing the compoundsand the enzyme in a similar manner as above (EcTopI relaxationinhibition assay) but in a gyrase assay buffer (35 mM Tris-HCl, 24 mMKCl, 4 mM MgCl₂, 2 mM DTT, 1.75 mM ATP, 5 mM spermidine, 0.1 mg/mL BSA,6.5% glycerol at pH 7.5), followed by the addition of 300 ng of relaxedcovalently closed circular DNA (New England Biolabs, Ipswich, Mass.,USA) to a final reaction volume of 20 μL. The samples were incubated at37° C. for 30 minutes before being terminated by the addition of abuffer containing 5% SDS, 0.25% bromophenol blue, and 25% glycerol. Thereactions were then analyzed by agarose gel electrophoresis.

Human Topoisomerase I Relaxation Inhibition Assay

Human topoisomerase I relaxation assays were carried out with 0.5 U ofenzyme in reaction buffer supplied by the manufacturer. The enzyme wasmixed with the indicated concentration of compound dissolved before 200ng of supercoiled pBAD/Thio plasmid DNA was added in the same buffer,for a final volume of 20 μL. Following incubation at 37° C. for 30minutes, the reactions were terminated with a buffer containing 5% SDS,0.25% bromophenol blue, and 25% glycerol, and analyzed by agarose gelelectrophoresis.

Human topoisomerase I and topoisomerase IIα were purchased from TopoGen(Buena vista, Colo., USA).

Human Topoisomerase IIα Decatenation Inhibition Assay

Human Topoisomerase IIα assays were carried out by adding the compoundsto 185 ng of kinetoplast DNA (kDNA, from TopoGen) in the buffer suppliedby the manufacturer before the addition of 2 U of the enzyme. Thesamples were incubated for 15 minutes at 37° C. before the addition of 4μL of a stop buffer containing 5% sarkosyl, 0.25% bromophenol blue, and25% glycerol. The reactions were then analyzed by electrophoresis in 1%agarose gels containing 0.5 μg/mL ethidium bromide before beingphotographed under UV light.

Selectivity and Specificity Against Other DNA Topoisomerase Enzymes

In addition, to determine the selectivity and specificity profiles ofthis class of fluoroquinophenoxazine derivatives, selected compoundswere also investigated for the ability to inhibit other DNAtopoisomerases including E. coli DNA gyrase as well as humantopoisomerase I and Ha enzymes.

Overall, these compounds were more selective toward E. colitopoisomerase I than other enzymes tested. Given that this series ofcompounds has close structural similarity to quinolone antibiotic class,as such, inhibition against E. coli gyrase can be also observed.Specifically, most of the compounds showed 4-64 fold selectivity towardtopoisomerase I over DNA gyrase except that the moderately activecompound 7e with the 9-NO₂ and 10-chloro substituents showed lessspecificity with 2 fold selectivity toward topoisomerase I. With respectto human topoisomerases I and IIα inhibition, these compounds alsoshowed inhibitory activity against both human topoisomerase I(IC₅₀=3.9-31.25 μM) and topoisomerase IIα (IC₅₀=0.97-250 μM), withapproximate 4-32 fold selectivity. Taken together, compounds IIα(IC₅₀=0.48 μM) and 11b (IC₅₀=0.24 μM) (FIG. 6) bearing both 9-NH₂ and6-piperazinyl motifs exhibited the most potent topoisomerase Iinhibitory activity with the more favorable selectivity profile (8-64fold) toward E. coli topoisomerase I against all the other enzymestested.

Example 5 Cell-Based Assays

The minimum inhibitory concentrations (MIC) of the compounds weredetermined against E. coli and B. subtilis in cation-adjustedMueller-Hinton Broth according to standard microdilution protocol [40].

MICs of compounds against M. tuberculosis were determined by a modifiedmicroplate Alamar blue assay (MABA) [41]. Vero cell cytotoxicity assaywas performed as previously described [41].

Cell-Based Antibacterial Activity

In addition to target-based topoisomerase enzyme inhibition, whole cellantibacterial activities of the synthesized compounds were also assessedagainst a panel of bacterial strains. The results are also shown inTable 1. From these data, the majority of these fluoroquinophenoxazinederivatives exhibited good to excellent antibacterial activity againstthe membrane permeable E. coli strain BAS3023 and Gram-positive B.subtilis strain, and were inactive against the wide type E. coli strain.Additionally, the antibacterial activity of most fluoroquinophenoxazinederivatives (e.g., 1, 7a, 7c, 9, 10a, 11a-d, 11f-i, 15a, 16, and 17a-b)generally correlated with E. coli topoisomerase I inhibitory activity,suggesting that the antibacterial basis of these compounds may be inpart due to the inhibition of topoisomerase I. The only type IAtopoisomerase present in M. tuberculosis has recently been validated asan antitubercular target [42]. The topoisomerase I activity has beenshown to be essential for viability and infection in a murine model oftuberculosis [42, 43]. To further determine the antituberculosis profilefor this chemical class of fluoroquinophenoxazine derivatives, twelvecompounds were selected and evaluated against M. tuberculosis. Amongthem, 11g with the 6-bipiperidinyl lipophilic side chain and 11d withthe 6-morpholino heterocyclic ring system showed the most potentantituberculosis activity with minimum inhibitory concentration (MIC)values of 2.5 and 3.5 μM, respectively. In addition, compared to 11d,its corresponding 6-piperazinyl structural analogs 11a with tertiaryamine and 11b with secondary amine functionality was about 2- and 8-foldless active with the MIC values of 7.6 and 29.5 μM, respectively. Incontrast, both 6-substituted aminopyrrolidinyl derivatives 17a (5) and17b (R) with primary amine functionality were not active (MIC>63.1 μM)against M. tuberculosis.

These data strongly suggest that the decreased or lost whole cellantituberculosis activity of these compounds are most likely due totheir decreased lipophilicity and subsequent cell membrane penetration.Unfortunately, cytotoxicity evaluation of our tested compounds againsthealthy normal Vero cells showed that they generally had narrowselectivity index, with 11g (SI=9.8) being the most promising compound.It is also worthwhile noting that, one of the most potent topoisomeraseI inhibitors, 11a (IC₅₀=0.48 μM) bearing the 6-methylpiperazinyl and9-amino motifs, showed broad spectrum antibacterial activity against allthe test bacteria strains with MICs ranging from 0.78 to 7.6 μM(SI=3.8-37).

TABLE 2 IC50 (μM) of compounds 11d, 11e, and 11g against Mycobacteriumtuberculosis topoisomerase I and MIC (μM) values of compounds 11d, 11e,and 11g for TB (Mycobacterium tuberculosis strain H37Rv) and MRSA(staphylococcus aureus BAA44). MIC MIC Code Structure IC50 (TB) (MRSA)11d

0.48 To be determined  3   11g

0.24  2.5 12.5 11e

0.24 21.6 25  

Viability of the mycobacteria mycobacterium smegmatis in biofilm wasmeasured by the resazurin assay. Compound 11 g at 1.5-3 μM can abolish90% of viability, versus 24 μM or higher required for ciprofloxacin toachieve the same anti-biofilm activity.

The resazurin assay is a quick method using the resazurin dye as abacterial respiration indicator to assay the antibacterial activity ofvarious compounds used against bacterial biofilm growth. Such assay iswell-known in the art. Resazurin was used to measure the presence ofactive biofilm bacteria, after adding the compound, in relation to astandard curve generated from inocula in suspension of the same organismused to grow the biofilm. The biofilm was quantified indirectly bymeasuring the fluorescent, water-soluble resorufin product produced whenresazurin is reduced by reactions associated with respiration.

Example 6 CoMFA Modeling Dataset

All the synthesized and tested fluoroquinophenoxazine derivatives wereused for CoMFA study. The topoisomerase I inhibitory activity (IC₅₀, μM)from biochemical enzyme assay was converted to pIC₅₀ values forcorrelation purpose (pIC₅₀=−logIC₅₀). The total compound set is dividedinto two subsets: a training set of 21 compounds for generating3D-quantitative structure-activity relationship (QSAR) models and a testset of 7 compounds for validating the quality of the model (Table 3).The compound selections of training and test sets were done manually sothat compounds ranging from weak, moderate, to strong topoisomerase Iinhibitory activities were present in both sets and were inapproximately equal proportions.

Conformational Model Analysis and Molecular Alignment

In the 3D-QSAR studies, alignment rule and biological conformationselection are two important factors to construct reliable models. Forboth training and test set molecules, conformational models representingtheir available conformational space were calculated. All the moleculeswere subjected to produce a maximum of 255 conformations within 20kcal/mol in energy from global minimum. Due to the relatively rigidstructural feature of these molecules, the core structure ofquinophenoxazine was used for the alignment.

CoMFA Model Generation

CoMFA was performed using the QSAR module of SYBYL-X [44]. The stericand electrostatic field energies were calculated using the Lennard-Jonesand the Coulomb potentials, respectively, with a 1/r distance-dependentdielectric constant in all intersections of a regularly spaced (0.2 nm)grid. The electrostatic fields were computed using Gasteiger-Huckelcharge calculation methods. A sp³ hybridized carbon atom with a radiusof 1.53 Å and a charge of +1.0 was used as a probe to calculate thesteric and electrostatic energies between the probe and the moleculesusing the Tripos force field. The standard parameters implemented inSYBYL-X were used. The truncation for both steric and electrostaticenergies was set to 30 kcal/mol.

Partial Least Square (PLS) Analysis

PLS methodology [45] was used for 3D-QSAR analysis. The cross-validationanalysis [46, 47] was performed using the leave one out (LOO) methods inwhich one compound is removed from the dataset and its activity is thenpredicted using the model derived from the rest of the dataset. Thecross validated r² that resulted in the optimum number of components andthe lowest standard error of prediction were considered for furtheranalysis. To speed up the analysis and reduce noise, a minimum filtervalue of 2.00 kcal/mol was used. A final analysis was performed tocalculate conventional r² using the optimum number of componentsobtained from the cross-validation analysis.

CoMFA Analysis

To further understand the structural basis for topoisomerase Iinhibitory activity of this set of fluoroquinophenoxazine derivatives,we subsequently performed three dimensional QSAR (3D-QSAR) study usingCoMFA analysis [48]. Because of the relatively rigid core structuralfeature of this class of fluoroquinophenoxazine molecules, we directlyapplied core structure based alignment to build reliable 3D-QSAR models.The CoMFA study was carried out using a total of 21 compounds (entries1-21, Table 3). Statistical parameters of the CoMFA model showed areasonable cross-validated correlation coefficient q² of 0.688,indicating a good internal prediction of the model. The CoMFA model alsoexhibited a conventional correlation coefficient r² of 0.806. Toevaluate the predictive ability of our developed model, a test set of 7compounds (entries 22-28, Table 3) which was not included in modelgeneration was subsequently used. The predicative correlationcoefficient r² _(pred) of 0.767 indicates good external predicativeability of the CoMFA model. The experimental and predicted values aswell as their residuals from the training and test set molecules arelisted in Table 3. The correlation between the predicted andexperimental values of all compounds was plotted and the resulting chartis shown in FIG. 7.

TABLE 3 Experimental (pIC₅₀) and CoMFA predicted activity (PA) valuesand residuals for the training and test set compounds^(a) Entry Compd.IC₅₀ (μM) pIC₅₀ CoMFA PA^(b) Δ^(c) 1 1 1.95 5.71 5.05 0.66 2  6b 31.254.51 4.21 0.30 3  6c 250 3.60 4.00 −0.40 4  7a 15.6 4.81 4.63 0.18 5  7b31.25 4.51 4.27 0.24 6  7c 250 3.61 4.15 −0.54 7  7e 31.25 4.51 4.54−0.03 8 9 1.95 5.71 5.88 −0.17 9 10a 1.95 5.71 6.05 −0.34 10 10b 3.95.41 5.90 −0.49 11 11a 0.48 6.32 6.21 0.11 12 11b 0.24 6.62 6.06 0.56 1311d 0.97 6.01 5.94 0.07 14 11e 3.9 5.41 5.83 −0.42 15 11f  5.85 5.235.39 −0.16 16 11g 0.48 6.31 6.15 0.16 17 11h 0.97 6.01 5.72 0.29 18 11k15.6 4.81 5.50 −0.69 19 15a 3.9 5.41 5.77 −0.36 20 16  0.48 6.32 5.880.44 21 17b 0.73 6.14 5.89 0.25 22  6a 15.6 4.81 4.55 0.26 23  7d 15.64.81 4.77 0.04 24 11c 3.9 5.41 5.76 −0.35 25 11i  3.9 5.41 5.23 0.18 2611j  3.9 5.41 5.45 −0.04 27 12  7.8 5.11 4.56 0.55 28 17a 0.73 6.14 5.950.19 ^(a)Entries 1-21 for training set; entries 22-28 for test set.^(b)Predicted activity. ^(c)Residual of experimental and predictedactivity values.

The results of the CoMFA model were analyzed and visualized using thestandard deviation coefficient (StDev*Coeff) mapping option contoured bysteric and electrostatic contributions. In order to probe thestructure/activity correlation, the steric and electrostatic contourswere mapped onto their aligned chemical structures of thesefluoroquinophenoxazine molecules to identify the potential regions inwhich the molecules would favorably or unfavorably interact with thetopoisomerase I enzyme. The representative steric and electrostaticcontour maps of the most active compound 11b and the least active 6cderived from the CoMFA model are shown in FIG. 5. Briefly, the yellowareas in the steric contour maps indicate regions of steric hindrance toactivity, while the green areas indicating steric contribution topotency. From the electrostatic contour maps, the regions in blueindicate positive electrostatic charge potential associated withincreased activity, with the red regions show electronegative groupswith increased activity.

In FIG. 8A and FIG. 8B, one green contour was found near the piperazinylmoiety of compound 11b indicating that a moderate steric substituentwould be favored at the 6 position of the quinophenoxazine scaffold.This may offer a potential explanation why the 6-substituted aminoderivatives were generally more active than the 6-fluoro analogs. Inaddition, two yellow contours were observed near the 9 position ofinactive 6c, indicating that a steric bulkiness (e.g., NO₂ in 6a and 7a,CF₃ in 6c, and the acetyl group in 7d) would be disfavored for activityin this area.

The CoMFA electrostatic contour maps are displayed in FIG. 8C and FIG.8D. A large blue contour was found around the 9 position of compounds11b and 6c, indicating that the presence of electron richfunctionalities and positively charged environment at this position(e.g., NH₂ vs. NO₂, Ac, CN, and CF₃) would be strongly favored fortopoisomerase I inhibitory activity. It was also observed that a big redcontour region was present around the 2 position offluoroquinophenoxazine scaffold, suggesting that an electronegativegroup (e.g., COOH and COOR₂) at this position may be required foractivity. Finally, two red contours were found at both sides of thefused heterocyclic skeleton of 11b and 6c, suggesting that electrondeficient functionalities would be favored in those regions.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes in light thereof willbe suggested to persons skilled in the art and are included within thespirit and purview of this application. In addition, any elements orlimitations of any invention or embodiment thereof disclosed herein canbe combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A compound having the following structure:

R₁ being selected from —OH, —NH₂, —NO₂, —NHMe, —Ac, —CN, —NHAc, —NHCH₂CH₂NH₂, —CF₃, fluorine, chloride, bromine, iodine, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocycloalkyl, and substituted heterocycloalkyl; R₂ being selected from H, alkyl, and substituted alkyl; R₃ being F; and R₄ being selected from fluorine, chloride, bromine, and iodine.
 2. The compound according to claim 1, R₁ being selected from —NH₂, —NHAc, —NO₂, —Ac, and —CF₃.
 3. The compound according to claim 1, R₂ being H or an ethyl group.
 4. The compound according to claim 1, R₄ being chloride.
 5. The compound according to claim 1, which is:


6. A pharmaceutical composition for treating a bacterial infection comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
 7. A method for treating a bacterial infection in a subject, the method comprising administering, to a subject in need of such treatment, an effective amount of a compound of claim
 1. 8. The method according to claim 7, the bacterial infection being caused by a bacterial pathogen selected from M. tuberculosis and non-tuberculosis mycobacteria (NTM).
 9. The method according to claim 7, the subject being a human.
 10. A method for inhibiting a bacterial topoisomerase, the method comprising administering, to a cell, an effective amount of a compound of claim
 1. 11. A compound having the following structure:

R₁ being selected from —NH₂, —NO₂, —Ac, —CN, —NHAc, and —CF₃; R₂ being selected from H, alkyl, and substituted alkyl; R₃ being selected from fluorine, chloride, bromine, iodine, amino,

R₄ being H, fluorine, chloride, bromine or iodine; and R₅ being H, —NH₂, —NO₂ or —NHBoc.
 12. The compound according to claim 11, R₂ being H or an ethyl group.
 13. The compound according to claim 11, R₃ being an amino group.
 14. The compound according to claim 11, R₄ being selected from fluorine, chloride, bromine and iodine.
 15. The compound according to claim 11, which is selected from:

R′ being selected from


16. A pharmaceutical composition for treating a bacterial infection comprising a compound of claim 11 and a pharmaceutically acceptable carrier.
 17. A method for treating a bacterial infection in a subject, the method comprising administering, to a subject in need of such treatment, an effective amount of a compound of claim
 11. 18. The method according to claim 17, the bacterial infection being caused by a bacterial pathogen selected from M. tuberculosis and non-tuberculosis mycobacteria (NTM).
 19. The method according to claim 17, the subject being a human.
 20. A method for inhibiting a bacterial topoisomerase, the method comprising administering, to a cell, an effective amount of a compound of claim
 11. 